PROJECT SUMMARY/ABSTRACT During cell division, chromosomes are duplicated and equally segregated into each daughter cell. The centromere, a specialized chromosomal structure, is responsible for correct segregation of chromosomes. The centromeres guide the assembly of the kinetochore, a multi-protein complex that links spindle microtubules to chromosomes during chromosome segregation. Mis-regulation of centromeres adversely affects chromosome segregation resulting in aneuploidy, or abnormal chromosome content, a condition found in more than 90% of all cancers. Centromeres are universally governed by the centromere-specific histone H3 variant, CENP-A. CENP-A replaces canonical histone H3 at centromeres, and provides the platform for the assembly of kinetochores. During replication of centromeric DNA, chromatin is disassembled ahead of the replication fork. Remarkably, after DNA replication, CENP-A is faithfully reassembled into nucleosomes of daughter centromeres but not elsewhere. How the parental CENP-A is faithfully recruited, i.e., inherited, to centromeric nucleosomes following DNA replication is completely unknown. In addition, mislocalization of CENP-A to non- centromeric regions has a devastating impact on chromosome segregation. How non-centromeric regions are protected from CENP-A mis-incorporation in normal cells also remains largely unexplored. Our long-term goal is to understand the molecular basis of the inheritance and specification of centromeres. Toward this goal, we propose to use fission yeast (Schizosaccharomyces pombe), a simple, genetically-tractable eukaryotic model organism. In fission yeast, many aspects of centromere regulation are evolutionarily conserved with humans. We have recently shown that proteins involved in DNA replication are required for faithful loading of CENP-A to centromeres. In Aim 1, we will test the hypothesis that DNA replication components interact with the CENP-A protein to propagate centromere assembly on newly replicated DNA in cells preparing for cell division. In addition, our preliminary results indicate that, like in multi-cellular organisms, overexpression of CENP-A in S. pombe results in chromosome mis-segregation and the assembly of CENP-A at non-centromeric chromatin. Using this system, we have demonstrated that the N-terminal domain of CENP-A plays a key role in preventing the incorporation of CENP-A at non-centromeric regions. In Aim 2, we will address the hypothesis that the N- terminal domain of CENP-A interacts with chromatin remodeling factors to protect non-centromeric chromatin from erroneously assembling CENP-A. In Aim 3, we will perform two novel complementary genome-wide genetic screens to identify factors involved in 1) promoting the assembly of endogenous CENP-A at centromeres and 2) protecting non-centromeric chromatin from assembling CENP-A. Further characterization of these factors will provide new insights into how CENP-A is precisely targeted to centromeric chromatin. The proposed research will shed light on the processes governing chromosome segregation in human cells, and hold promise for a better understanding of cancer progression and the development of new cancer treatments.